Spectroscopic studies of quinonoid species from pyridoxal 5'-phosphate

Metzler, Carol M.; Harris, A. G.; Metzler, David E.

doi:10.1021/bi00413a050

Cited by 44 publications

(52 citation statements)

References 37 publications

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“…Global analysis of the multiwavelength data provides the spectra for the three species in the biphasic process. The spectra of species A have clear absorbance at 495 nm, which is characteristic of a quinonoid intermediate (16). Also, the spectra of species A show a hyperbolic dependence on alanine concentration, similar to that observed with AIB.…”

Section: Dgd-plp Transamination Half-reaction With L-alaninesupporting

confidence: 72%

“…Multiwavelength data show a decrease in the absorbance at 410 nm, concomitant with an increase in absorbance at 330 nm and a shift in the λ max of the remaining aldehydic absorbance to 395 nm. Global analysis of the multiwavelength data using a two-step, serial mechanism (eq 1) in SPECFIT gives spectra for three species (A, B, and C; Figure 2A) An isosbestic point at 352 nm is observed with no absorbance at ∼500 nm, characteristic of a quinonoid intermediate (16). The observed rate constants from the global fits increase hyperbolically with AIB concentration, with apparent y-intercepts of zero.…”

Section: Heats Of Activationmentioning

confidence: 99%

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Rapid Kinetic and Isotopic Studies on Dialkylglycine Decarboxylase

et al. 2001

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The two half-reactions of the pyridoxal 5′-phosphate (PLP)-dependent enzyme dialkylglycine decarboxylase (DGD) were studied individually by multiwavelength stopped-flow spectroscopy. Biphasic behavior was found for the reactions of DGD-PLP, consistent with two coexisting conformations observed in steady-state kinetics [Zhou, X., and Toney, M. D. (1998) Biochemistry 37, 5761-5769]. The halfreaction kinetic parameters depend on alkali metal ion size in a manner similar to that observed for steadystate kinetic parameters. The fast phase maximal rate constant for the 2-aminoisobutyrate (AIB) decarboxylation half-reaction with the potassium form of DGD-PLP is 25 s -1 , while that for the transamination half-reaction between DGD-PMP and pyruvate is 75 s -1 . The maximal rate constant for the transamination half-reaction of the potassium form of DGD-PLP with L-alanine is 24 s -1 . The spectral data indicate that external aldimine formation with either AIB or L-alanine and DGD-PLP is a rapid equilibrium process, as is ketimine formation from DGD-PMP and pyruvate. Absorption ascribable to the quinonoid intermediate is not observed in the AIB decarboxylation half-reaction, but is observed in the dead-time of the stopped-flow in the L-alanine transamination half-reaction. The [1-13 C]AIB kinetic isotope effect (KIE) on k cat for the steady-state reaction is 1.043 ( 0.003, while a value of 1.042 ( 0.009 was measured for the AIB half-reaction. The secondary KIE measured for the AIB decarboxylation halfreaction with [C4′-2 H]PLP is 0.92 ( 0.02. The primary [2-2 H]-L-alanine KIE on the transamination halfreaction is unity. Small but significant solvent KIEs are observed on k cat and k cat /K M for both substrates, and the proton inventories are linear in each case. NMR measurements of C2-H washout vs product formation give ratios of 105 and 14 with L-alanine and isopropylamine as substrates, respectively. These results support a rate-limiting, concerted CR-decarboxylation/C4′-protonation mechanism for the AIB decarboxylation reaction, and rapid equilibrium quinonoid formation followed by rate-limiting protonation to the ketimine intermediate for the L-alanine transamination half-reaction. Energy profiles for the two half-reactions are constructed.Dialkylglycine decarboxylase (DGD) 1 is an intriguing PLP-dependent enzyme because it catalyzes both transamination and decarboxylation reactions in its normal catalytic cycle (Scheme 1). The enzyme was isolated from a soil bacterium (1) and transfers the fixed nitrogen from small 2,2-dialkylglycines, which are common in peptide antibiotics (2), onto pyruvate to give L-alanine. The decarboxylation half-reaction is highly specific for oxidative decarboxylation. Thus, the issue of reaction specificity has two main facets with this enzyme: control of decarboxylation vs transamination, and control of oxidative vs nonoxidative decarboxylation. The latter is addressed in this work.Previous work has shown that DGD activity is dependent on alkali metal ions; potassium activates it w...

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Section: Dgd-plp Transamination Half-reaction With L-alaninesupporting

confidence: 72%

Section: Heats Of Activationmentioning

confidence: 99%

Rapid Kinetic and Isotopic Studies on Dialkylglycine Decarboxylase

et al. 2001

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“…3). Evidence that the presence of a proton or methyl group on N-1 of PLP facilitates quinonoid formation is provided by studies of quinonoid formation in model and enzymatic systems with pyridoxal and N-methylpyridoxal (41). Investigations of the spectra of quinonoids derived from O-methyl-PLP and PLP suggest that the proton may have migrated from the Schiff base nitrogen to O-3Ј (41,42), as shown in the structure in Fig.…”

Section: Table Imentioning

confidence: 99%

Mutation of an Active Site Residue of Tryptophan Synthase (β-Serine 377) Alters Cofactor Chemistry

Jhee

Yang²,

Ahmed³

et al. 1998

Journal of Biological Chemistry

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To better understand how an enzyme controls cofactor chemistry, we have changed a tryptophan synthase residue that interacts with the pyridine nitrogen of the pyridoxal phosphate cofactor from a neutral Ser (␤-Ser 377 ) to a negatively charged Asp or Glu. The spectroscopic properties of the mutant enzymes are altered and become similar to those of tryptophanase and aspartate aminotransferase, enzymes in which an Asp residue interacts with the pyridine nitrogen of pyridoxal phosphate. The absorption spectrum of each mutant enzyme undergoes a pH-dependent change (pK a ϳ ϳ 7.7) from a form with a protonated internal aldimine nitrogen ( max ‫؍‬ 416 nm) to a deprotonated form ( max ‫؍‬ 336 nm), whereas the absorption spectra of the wild type tryptophan synthase ␤ 2 subunit and ␣ 2 ␤ 2 complex are pHindependent. The reaction of the S377D ␣ 2 ␤ 2 complex with L-serine, L-tryptophan, and other substrates results in the accumulation of pronounced absorption bands ( max ‫؍‬ 498 -510 nm) ascribed to quinonoid intermediates. We propose that the engineered Asp or Glu residue changes the cofactor chemistry by stabilizing the protonated pyridine nitrogen of pyridoxal phosphate, reducing the pK a of the internal aldimine nitrogen and promoting formation of quinonoid intermediates.An important question in investigations of enzyme structure and function is how enzymes have evolved different reaction and substrate specificities. Pyridoxal phosphate (PLP) 1 -dependent enzymes are attractive targets for addressing this question because they catalyze a wide variety of reactions of amino acids (1, 2). The enzyme protein directs and restricts the catalytic potential of the bound PLP to provide the substrate and reaction specificity and the enhanced reaction rate of the enzyme (3, 4). Thus, it is important to understand how the protein structure controls the cofactor chemistry and enhances catalytic rates.Information on how the protein structure controls PLP-dependent reactions is beginning to emerge from x-ray crystallography and from sequence comparisons designed to establish evolutionary relationships (5, 6). One of the most important and best studied interactions between the cofactor and the protein active site of all PLP enzymes is that between the pyridine nitrogen of PLP and an amino acid side chain (see Fig.

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“…Condensation of THT-A with enzyme-bound PLP forms an aldimine intermediate. Loss of the ␣-proton of the amino acid produces an intermediate described as a quinonoid-carbanion structure (Metzler et al, 1988). Extending the conjugation of the system by elimination of tetrahydrothiophene is likely to be a process that is favored over imine hydrolysis.…”

Section: Plp-catalyzed Formation Of Pyruvate and Ammonium From Tht-amentioning

confidence: 99%

Metabolism of the CysteineS-Conjugate of Busulfan Involves a β-Lyase Reaction

Cooper

Younis²,

Niatsetskaya³

et al. 2008

Drug Metab Dispos

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ABSTRACT:The present work documents the first example of an enzymecatalyzed ␤-elimination of a thioether from a sulfonium cysteine S-conjugate. ␤-(S-Tetrahydrothiophenium)-L-alanine (THT-A) is the cysteine S-conjugate of busulfan. THT-A slowly undergoes a nonenzymatic ␤-elimination reaction at pH 7.4 and 37°C to yield tetrahydrothiophene, pyruvate, and ammonia. This reaction is accelerated by 1) rat liver, kidney, and brain homogenates, 2) isolated rat liver mitochondria, and 3) pyridoxal 5-phosphate (PLP). A PLPdependent enzyme in rat liver cytosol that catalyzes a ␤-lyase reaction with THT-A was identified as cystathionine ␥-lyase. This unusual drug metabolism pathway represents an alternate route for intermediates in the mercapturate pathway.

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Spectroscopic studies of quinonoid species from pyridoxal 5'-phosphate

Cited by 44 publications

References 37 publications

Rapid Kinetic and Isotopic Studies on Dialkylglycine Decarboxylase

Rapid Kinetic and Isotopic Studies on Dialkylglycine Decarboxylase

Mutation of an Active Site Residue of Tryptophan Synthase (β-Serine 377) Alters Cofactor Chemistry

Metabolism of the CysteineS-Conjugate of Busulfan Involves a β-Lyase Reaction

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